Opioid analgesic drugs modulate primary nociceptive neurons by activating mu (MOR) and delta (DOR) - type G protein-coupled opioid receptors. The beta-2 adrenergic receptor (B2AR) is a distinct G protein- coupled receptor expressed in nociceptive neurons that can produce enhanced, rather than reduced, nociceptive responses. Studies of heterologous cell models indicate that both opioid and adrenergic receptors are regulated by a shared mechanism, involving receptor phosphorylation by G protein-coupled receptor kinases (GRKs) followed by rapid endocytosis. Little is known about the occurrence or function of this conserved regulatory mechanism in nociceptive neurons. A combination of immunohistochemical and live cell imaging approaches will be used to investigate the steady state distribution and regulated endocytic trafficking of MOR, DOR and B2AR in nociceptive neurons, both in the intact tissue and in dissociated neuronal culture. The effects of selected opioid and adrenergic agonists on regulated endocytosis of receptors will be determined using quantitative trafficking assays. The possible functional importance of a specific mechanism of receptor sorting after endocytosis, which determines the signaling consequences of regulated endocytosis in heterologous cell models, will be probed in nociceptive neurons using previously established mutations. A novel protein engineering method will be applied to temporally control GRK activity in nociceptive neurons, and to develop a new approach potentially suitable for probing functional effects on nociceptive signaling in vivo. These studies will provide fundamental insight to cell biological mechanisms relevant to opioid analgesia and tolerance. They may also provide insight to mechanisms that modulate hyperalgesia produced by adrenergic activation in nociceptive neurons.